Biocatalytic processes have become very important to the chemical industry. Of particular importance is the use of enzymes, when the properties of biocatalysts enable either of the two enantiomers in chemical reaction with chiral or prochiral compounds to be reacted or formed preferentially.
Essential requirements for utilizing these favorable properties of enzymes are their low-cost availability in sufficient amounts, as required in industrial processes, a sufficiently high reactivity, selectivity and high stability under the realistic conditions of the industrial process.
β-nitro alcohols are precursors for β-amino alcohols, which are important chiral building blocks for the synthesis of bioactive compounds, such as ephedrine, bestatin and sphingosine, used as pharmaceutical ingredients. The nitroaldol or Henry reaction is one of the classical named reactions in organic synthesis for C—C bond formation. Due to the potential to create up to two new chiral centers it is of fundamental importance for synthetic applications to be able to perform the nitroaldol addition enantio- and stereoselectively. Although the reaction has been known for more than a century (Henry, 1895), stereospecific protocols utilizing non-enzymatic organocatalysts or chiral metal catalysts have been developed only recently. The development of these methods is impressive, but they still share a number of disadvantages, including long reaction times and sometimes extreme reaction conditions in the case of metal catalysts, or insufficient selectivities in the case of organocatalysts.
In the past decade, the first asymmetric biocatalytic nitroaldol reaction was discovered for the hydroxynitrile lyase from the tropical rubber tree Hevea brasthensis (HbHNL) (Purkarthofer et al., Angew Chem Int Ed Engl. 2006 45(21):3454-6, Gruber-Khadjawi et al., Adv. Synth. Catal. 2007, 349, 1445-1450, Yuryev, R.; et al., Biocatal. Biotransform., (2010) 28, 348; Yuryev, R.; et al.; Chemcatchem, (2010) 2, 981)).
The (S)-selective MeHNL from Manihot esculents, which like HbHNL belongs to the α/β-hydrolase superfamily, is also capable of catalyzing the (S)-selective nitroaldol reaction, albeit with lower activity and selectivity.
The first (R)-selective HNL, which catalyzes the (R)-selective HNL-catalyzed Henry reaction is AtHNL from Arabidobsis thaliana (Fuhshuku et al. J. Biotechnol. 2011, 153, 153-159), which belongs also to the α/β-hydrolase superfamily like the (S)-selective nitroaldolases.
In contrast, activity in the nitroaldol reaction has not been shown so far for the (R)-selective hydroxynitrile lyase from Prunus amygdalus (PaHNL), which belongs to a different protein fold.
Unfortunately, however, the enantiomeric excess of the reaction product of AtHNL decreases significantly without increase of yield during prolonged reaction times (from 2 h to 4 h) at the reported reaction conditions (Fuhshuku et al. 2011).
Asano and coworkers achieved the highest enantioselectivity for benzaldehyde and MeNO2 in a biphasic system at pH 7 with 50% n-butyl acetate (30% yield and 91% ee) applying 40 mg AtHNL per mmol benzaldehyde. Yield and enantiomeric excess were further slightly improved by applying larger amounts of enzyme (100 mg per mmol of substrate). Depending on the substrate and reaction system yields up to 60% or enantiomeric excess up to 96% could be obtained applying 40 mg of AtHNL per mmol of substrate and a reaction time of 2 h. However, not coexistent under the same reaction conditions.
However, the enantiomeric excess of the reaction product of AtHNL decreases significantly without increase of yield during prolonged reaction times (from 2 h to 4 h) at the reported reaction conditions. Nitroethane was not used.
Gotor and coworkers reported the protein-mediated catalysis of the nitroaldol reaction by the carrier protein bovine serum albumin (BSA) in water, which can be categorized as organocatalysis because the observations of the scientists led to the conclusion of unspecific protein catalysis. (Busto, E.; Gotor-Fernandez, V.; Gotor, V., Org. Process Res. Dev., (2011) 15, 236). Biocatalytic nitroaldol reactions were also reported with enzymes (for a review see Milner, S. E.; et al., Eur. J. Org. Chem, (2012), 3059), such as a transglutaminase, (Tang, R. C.; et al., J. Mol. Catal. B: Enzym., (2010) 63, 62) a hydrolase, (Wang, J. L.; et al., J. Biotechnol., (2010) 145, 240) a protease, (Lopez-Iglesias, M.; et al., Adv. Synth. Catal., (2011) 353, 2345) lipases, (Le, Z.-G.; et al., Green Chem. Lett. Rev., (2013) 6, 277; Xia, W.-J.; et al., Molecules, (2013) 18, 13910), an acylase (Xia, W.-J.; et al., Molecules, (2013) 18, 13910) and a glucoamylase (Gao, N.; et al., RSC Adv., (2013) 3, 16850), but in all cases the reactions were not enantioselective or no data about enantioselectivity were provided.
Thus, so far only plant HNLs with α/β-hydrolase fold were capable of catalysing the enantioselective nitroaldol reaction.
Another approach is a chemo-enzymatic approach, in which the chemically synthesized mixture of stereoisomers is separated by enzymatic kinetic resolution, e.g. using hydrolases. However, the major drawback of kinetic resolution in general is the limitation of the yield to a maximum of 50%.
Thus, there is still the need for new nitroaldolases, which can enantioselectively catalyze the Henry reaction.
Recently, the discovery of new bacterial HNLs with cupin fold has been reported (Hajnal, I.; et al., Febs J., (2013) 280, 5815; Hussain, Z.; et al., Appl. Environ. Biotechnol., (2012) 78, 2053), however displaying only very low specific activity. One of the new enzymes, GtHNL, was characterized in detail and its structure was solved (Hajnal, I.; et al., Febs J., (2013) 280, 5815; Lyskowski, A.; et al., Acta Crystallogr. F, (2012) 68, 451). It is a small metal-dependent mono-cupin with a molecular weight of ˜15 kDa, which forms a tetramer.